1. Technical Field
The present invention is directed to proteins, nucleic acid sequences and constructs, and methods related thereto.
2. Background
Fatty acids are organic acids having a hydrocarbon chain of from about 4 to 24 carbons. Many different kinds of fatty acids are known which differ from each other in chain length, and in the presence, number and position of double bonds. In cells, fatty acids typically exist in covalently bound forms, the carboxyl portion being referred to as a fatty acyl group. The chain length and degree of saturation of these molecules is often depicted by the formula CX:Y, where "X" indicates number of carbons and "Y" indicates number of double bonds.
The production of fatty acids in plants begins in the plastid with the reaction between acetyl-CoA and malonyl-ACP to produce butyryl-ACP catalyzed by the enzyme, .beta.-ketoacyl-ACP synthase III. Elongation of acetyl-ACP to 16- and 18-carbon fatty acids involves the cyclical action of the following sequence of reactions: condensation with a two-carbon unit from malonyl-ACP to form a .beta.-ketoacyl-ACP (.beta.-ketoacyl-ACP synthase), reduction of the keto-function to an alcohol (.beta.-ketoacyl-ACP reductase), dehydration to form an enoyl-ACP (.beta.-hydroxyacyl-ACP dehydrase), and finally reduction of the enoyl-ACP to form the elongated saturated acyl-ACP (enoyl-ACP reductase). .beta.-ketoacyl-ACP synthase I, catalyzes elongation up to palmitoyl-ACP (C16:0), whereas .beta.-ketoacyl-ACP synthase II catalyzes the final elongation to stearoyl-ACP (C18:0). The longest chain fatty acids produced by the FAS are typically 18 carbons long. A further fatty acid biochemical step occurring in the plastid is the desaturation of stearoyl-ACP (C18:0) to form oleoyl-ACP (C18:1) in a reaction catalyzed by a .DELTA.-9 desaturase, also often referred to as a "stearoyl-ACP desaturase" because of its high activity toward stearate the 18 carbon acyl-ACP.
Carbon-chain elongation in the plastids can be terminated by transfer of the acyl group to glycerol 3-phosphate, with the resulting glycerolipid retained in the plastidial, "prokaryotic", lipid biosynthesis pathway. Alternatively, specific thioesterases can intercept the prokaryotic pathway by hydrolyzing the newly produced acyl-ACPs into free fatty acids and ACP.
Subsequently, the free fatty acids are converted to fatty acyl-CoA's in the plastid envelope and exported to the cytoplasm. There, they are incorporated into the "eukaryotic" lipid biosynthesis pathway in the endoplasmic reticulum which is responsible for the formation of phospholipids, triglycerides and other neutral lipids. Following transport of fatty acyl CoA's to the endoplasmic reticulum, subsequent sequential steps for triglyceride production can occur. For example, polyunsaturated fatty acyl groups such as linoleoyl and a-linolenoyl, are produced as the result of sequential desaturation of oleoyl acyl groups by the action of membrane-bound enzymes. Triglycerides are formed by action of the 1-, 2-, and 3-acyl-ACP transferase enzymes glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase and diacylglycerol acyltransferase. The fatty acid composition of a plant cell is a reflection of the free fatty acid pool and the fatty acids (fatty acyl groups) incorporated into triglycerides as a result of the acyltransferase activities.
The properties of a given triglyceride will depend upon the various combinations of fatty acyl groups in the different positions in the triglyceride molecule. For example, if the fatty acyl groups are mostly saturated fatty acids, then the triglyceride will be solid at room temperature. In general, however, vegetable oils tend to be mixtures of different triglycerides. The triglyceride oil properties are therefore a result of the combination of triglycerides which make up the oil, which are in turn influenced by their respective fatty acyl compositions.
Plant acyl-ace carrier protein thioesterases are of biochemical interest because of their roles in fatty acid synthesis and their utilities in bioengineering of plant oil seeds. A medium-chain acyl-ACP thioesterase from California bay tree, Umbellularia californica, has been isolated (Davies et al. (1991) Arch. Biochem. Biophys. 290:37-45), and its cDNA cloned and expressed in E.coli (Voelker et al. (1994) J. Bacterial. 176:7320-7327) and seeds of Arabidopsis thaliana and Brassica napus (Voelker et al. (1992) Science 257:72-74). In all cases, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. These results demonstrated the role of the TE in determining chain-length during de novo fatty acid biosynthesis in plants and the utility of these enzymes for modifying seed oil compositions in higher plants.
Recently, a number of cDNA encoding different plant acyl-ACP thioesterases have been cloned (Knutzon et al. (1992) Plant Physiol. 100:1751-1758; Voelker, et al. (1992) supra; Dormann et al. (1993) Planta 189:425-432; Dormann et al. (1994) Biochim. Biophys. Acta 1212:134-136; Jones et al. (1995) The Plant Cell 7:359-371). Sequence analyses of these thioesterases show high homology, implying similarity in structure and function. Some of these thioesterase cDNAs have been expressed in E.coli, and their substrate specificities determined by in vitro assays. The fact that these enzymes share significant sequence homology, yet show different substrate specificities, indicates that subtle changes of amino acids may be sufficient to change substrate selectivity.
Little information is available on structural and functional divergence amongst these plant thioesterases, and the tertiary structure of any plant thioesterase has yet to be determined. Protein engineering may prove to be a powerful tool for understanding the mechanism of thioesterase substrate recognition and catalysis, and thus lead to the rational design of new enzymes with desirable substrate specificities. Such new enzymes would find use in plant bioengineering to provide various modifications of fatty acyl compositions, particularly with respect to production of vegetable oils having significant proportions of desired fatty acyl groups, including medium-chain fatty acyl groups (C8 to C14) and longer chain fatty acyl groups (C16 or C18). In addition, it is desirable to control the relative proportions of various fatty acyl groups that are present in the seed storage oil to provide a variety of oils for a wide range of applications.
Literature
The strategy of using chimeric gene products has been applied to study the structure and function of phosphotransferases in yeast (Hjelmstad et al. (1994) J. Biol. Chem. 269: 20995-21002) and restriction endonucleases of Flavobacterium Kim et al. (1994) Proc. Natl. Acad. Sci. USA. 91:883-887).
Domain swapping to rearrange functional domains of proteins has been used in protein engineering (Hedstrom (1994) Current Opinion in Structural Biology 4:608-611). Recently the structure of a myristoyl-ACP thioesterase from Vibrio harveyi has been determined (Lawson et al. (1994) Biochemistry 33:9382-9388). This thioesterase, like other bacterial or mammalian thioesterases, shares no sequence homology with plant thioesterases (Voelker et al. (1992) supra).